The molecular mechanisms involved in DNA replication are being studied biochemically. In particular, in vitro replication reactions are being used to study origin specific initiation of replication. I have developed an in vitro DNA replication system that replicates exogenously added plasmid DNA containing the origin of replication of bacteriophage P1. The system consists of a purified P1 replication protein, the product of repA, and a partially purified mixture of E. coli replication proteins prepared from uninfected cells. The system requires the E. coli dnaA initiation protein in addition to the P1 RepA initiation protein. In collaboration with D. Chattoraj (LB, NCI), we have shown by electron microscopic techniques that replication is initiated in the region of the P1 origin of replication and proceeds undirectionally. Since P1 normally exists as a unit copy plasmid, this system is being used to study the molecular mechanisms involved in the initiation and regulation of a stringently controlled replicon. I have also continued studying the replication of plasmid DNA containing the origin of replication of bacteriophage lambda in vitro. This reaction requires two phage functions, the O and P gene products, many host replication proteins and several other host proteins including the heat shock proteins, dnaJ, dnaK, and grpE. Replication also requires a specific DNA site for initiation. I have constructed deletions in vitro extending into this region and tested them for activity in in vitro DNA replication reactions dependent on O and P proteins. The smallest piece of DNA that supports the initiation of replication is 89 bp. It contains two of the four O protein binding sites and the adjoining adenine and thymine rich region that very likely is the site where dnaB protein is transferred to the DNA. In a collaborative electron microscopic study with M. Dodson and H. Echols (University of CA, Berkeley), nucleoprotein structures formed by O, P, dnaB, dnaJ, dnaK, and Ssb at ori lambda have been visualized.